Abstract: Synapse loss induced by HIV-1 proteins in the presence of ART and drugs of abuse HIV-1 associated neurocognitive disorder (HAND) affects almost half of infected individuals. Cognitive impairment in these patients correlates with synaptodendritic damage. To study HIV-1 associated synaptic changes, a high content analysis (HCA) assay was developed to quantify functional glutamatergic synapses between hippocampal neurons in culture. This highly automated assay is exquisitely sensitive to HIV-1 proteins and neuroinflammation. In this proposal, the use of the HCA assay to examine the concentration- and time-dependent synaptic toxicity of HIV-1 proteins, antiretroviral drugs, and drugs of abuse, alone and in combination is described. The first aim will determine whether HIV-1 proteins in combination synergize to increase synaptic toxicity. Taking advantage of the sensitivity, throughput and quantitative rigor of the HCA assay all HIV-1 proteins will be tested singly and in combinations. Concentration response data will enable identification of antagonistic, additive, and synergistic synaptic toxicities produced by combining the various proteins. The second aim will determine whether antiretroviral drugs produce synaptic toxicity. There is growing concern that combined antiretroviral therapy (cART) may elicit some direct neurotoxicity. With more than 20 antiretroviral drugs approved by the FDA for HIV-1 and the expectation that ART-induced neurotoxicity might be subtle, a comprehensive assessment of their potential neurotoxicity, particularly in the presence of HIV-1 neurotoxins, is lacking. All FDA approved ART drugs and ART combinations will be tested for synaptic toxicity at clinically relevant concentrations. These results will identify ART drugs with a propensity to impair synaptic function. The third aim will determine whether drugs of abuse produce synaptic toxicity. 40% of HIV- 1 infected individuals used illegal drugs within the past year and substance abuse is a risk factor for neurological dysfunction in these patients. A comprehensive survey of how drugs of abuse influence the toxicity of HIV-1 proteins and ART drugs in combination is lacking. Prototype drugs representing major classes of abused drugs will be tested for synaptic toxicity at concentrations detected in the brain at intoxicating doses. The fourth aim will evaluate HIV-1 protein and drug combinations for synaptic toxicity. The most potent agents identified in Aims 1-3 will be tested in combination. The goal is to identify combinations of HIV-1 proteins, ART drugs, and drugs of abuse that induce synapse loss with high potency. Neurotoxic combinations will be evaluated for synergistic effects, desensitizing effects following prior exposure to drugs of abuse, and effects of prolonged treatment. The anticipated results will establish the feasibility of using HCA assays to identify synergistic CNS toxicities from combinations of neuroinflammatory agents. These studies will provide a foundation for future studies of the mechanism and potential reversal of synapse loss. Newly identified toxicities will be validated with follow up in vivo studies and could inform clinical care of HAND patients.